Sepsis is the leading cause of death among critically ill patients in the United States with between 230,000 and 370,000 people dying from the disease annually. Outside of antibiotics, treatment for sepsis is non-specific, and there are no approved therapeutics available once antibiotics and supportive therapy fail. The gut has long been characterized as the motor of multiple organ dysfunction syndrome. We have spent the three previous cycles of funding examining mechanisms of gut integrity (first apoptosis then proliferation, migration and permeability) in sepsis. This renewal is a logical next step in the evolution of how we view the gut in sepsis. We propose to assay the intestinal epithelium, immune system and microbiome, both in isolation and also in the context of how alterations in one compartment impact the others since we hypothesize this strategy will yield insights that can only be obtained using this more comprehensive approach. The first goal of the proposal is to understand mechanisms through which the immune system and microbiome alter survival in mice lacking the tight junction-associated protein junctional adhesion molecule-A (JAM-A), which have alterations in permeability, bacteremia and survival following sepsis. This will be done using a combination of mice with whole body and intestine-specific deletion of JAM-A as well as mice with controlled alterations in the endogenous bacteria. Further, intestinal permeability is controlled by two tight junction-dependent pathways and a tight junction-independent pathway. Each allows different size molecules to exit the gut lumen into the extraluminal environment. By genetically altering each of these pathways of permeability (the leak, pore and unrestricted pathways respectively), studies will determine the functional significance of each together and in isolation in sepsis. Finally, migration is slowed along the intestine during sepsis, with cells residing nearly twice as long during sepsis as under basal conditions, mediated, at least in part, by apoptosis and proliferation. Mechanisms of slowed migration will be determined including the impact of altering permeability and the microbiome. Since the gut plays a major role in both initiating and propagating critical illness, understanding mechanisms through which gut integrity is dysregulated in sepsis has significant public health implications in a disease that is common, very costly, and highly lethal.